Control of the cell cycle is fundamental to the growth and maintenance of eukaryotic organisms, from yeasts to mammals. Cells have evolved critical mechanisms to help protect the fidelity of DNA synthesis. One important mechanism is commonly referred to as “cell-cycle checkpoint control”. Cell cycle checkpoints insure that individual steps of the cell cycle are completed before the next step occurs. In response to DNA damage or a block to DNA replication, progression through the cell cycle is delayed. This allows time for the cell to repair the DNA prior to continuing through the cell cycle, thus improving genomic stability and the fidelity of DNA synthesis (Elledge (1996) Science 274: 1664–1672; O'Connell et al. (2000) Trends Cell Biol 10: 296–303).
The ability to coordinate cell cycle transitions in response to genotoxic and other stressors is critical to the maintenance of genetic stability and the prevention of uncontrolled cellular growth. Loss of a checkpoint gene leads to genetic instability and the inability of cells to deal with genomic insults such as those suffered as a result of the daily exposure to ultraviolet radiation. The loss of negative growth control and improper monitoring of the fidelity of DNA replication are common features of tumor cells. When checkpoints are eliminated (e.g., by mutation or other means), cell death, infidelity in chromosome transmission, and/or increased susceptibility to deleterious environmental factors (e.g., DNA-damaging agents) result.
Many components of the checkpoint pathways that respond to DNA damage have been identified in various species from yeast to vertebrates (Elledge (1996) Science 274: 1664–1672). The response is believed to involve sensor proteins which respond to DNA damage and/or replication stress. The sensor proteins transmit a signal (via transducer proteins) which induces one or more effects in a cell. Such effects allow the cell to appropriately cope with the DNA damage by, for example, inducing a cell cycle delay to allow time for the DNA damage to be repaired. Other possible responses of a cell to DNA damage include cell death, for example, if the DNA damage is too great to be repaired (recently reviewed in Zhou and Elledge (2000) Nature 408: 433–439).
One class of sensor proteins include Rad3/ATR proteins (Bentley et al. (1996) EMBO Journal 15: 6641–6651; O'Connell et al. (2000) Trends Cell Biol 10: 296–303; Cimprich et al. (1996) PNAS 93: 2850–2855; Keegan et al. (1996) Genes & Development 10: 2423–2437). This family of sensor proteins actually is part of a larger family of phosphoinositide kinase (PIK)-related protein kinases. This family of PIK-kinases are characterized by a C-terminal kinase domain and include ATM/Tell (Lavin and Shiloh (1997) Annu. Rev Immunology 15: 177–202; Sanchez et al. (1996) Science 271: 357–360) and DNA-PKcs (Smith and Jackson (1999) Genes & Development 13: 916–934).
Following detection of DNA damage or a replication block, a signal is transduced to effector proteins. These include Chk1 and Cds1 (Elledge (1996) Science 274: 1664–1672). However, the molecular nature of how this signal is transduced is not well understood. Based on previous work, it appears that various sensors induce cell cycle delay in response to different types of DNA damage, and that different sensors signal through different effector proteins. Additionally, extensive variability has been observed in the results obtained across species. Thus, although it appears that the general machinery for checkpoint control in response to DNA damage is evolutionarily conserved, it has remained uncertain as to whether the specific molecular mechanisms employed to accomplish these goals are also conserved.
Given the importance of proper checkpoint control in maintaining genomic stability and insuring the fidelity of DNA replication, a better understanding of the molecular mechanisms underlying this process has tremendous value. Specifically, such an understanding allows for the development of rational screens for agents which can modulate checkpoint control in response to DNA damage. Such agents provide novel therapies for various proliferative disorders including all forms of cancer.
The present invention aims to address the shortcomings of the prior art. We describe the isolation and characterization of Xenopus ATR nucleic acids and proteins. The characterization of Xenopus ATR revealed insights into a specific mechanism whereby DNA damage is sensed and then transduced to induce a cell cycle delay. The present invention demonstrates that ATR phosphorylates Chk1 (e.g., Chk1 is a substrate for the ATR kinase), and that this phosphorylation is an evolutionarily conserved mechanism necessary for the cell cycle delay induced by DNA damage or a DNA replication block.
The teachings of the present invention allow, for the first time, methods of screening for agents which modulate the activity of an ATR protein in any species. Such screens will not only increase understanding of cell cycle checkpoints, but will also provide possible therapeutic agents for the treatment of proliferative disorders.